The availability of large neutral amino acids in brain cells is crucial to the regulation of both cerebral protein metabolism and neurotransmitter synthesis. The transport of circulating amino acids from blood to brain involves transport through two membranes in series: (i) the brain capillary endothelial wall, which makes up the blood-brain barrier (BBB) in vivo, and (ii) the brain cell (neuron, glial) plasma membrane. Since the surface area of the brain cell membrane is log orders greater than the surface area of the blood-brain barrier, the rate limiting step in amino acid movement from plasma to brain intracellular space is the BBB transport step. This work will study the pathophysiological expression of the large neutral amino acid transporter (LAT) at the blood-brain barrier. The preliminary data show that the full length LAT-cDNA encodes a protein that expresses LAT activity in frog oocytes. Antisera will be produced with synthetic peptides encoding either the carboxyl terminus or predicted extracellular domains of the bovine BBB LAT. The abundance and cellular localization of this transporter will be studied by Western blotting, ELISA, in situ hybridization, immunocytochemistry, and confocal and immunogold electron microscopy, respectively. The modulation of gene expression of BBB LAT will be studied under different pathophysiologic conditions known to modify the transporter activity, i.e. brain tumors, development, hypothyroidism and hypoxia. The mechanisms of gene regulation of BBB LAT will be investigated in brain endothelial cultured cells measuring the abundance of its protein and transcript, and the transcriptional rate and decay of the BBB LAT mRNA. The cloning of cDNAs encoding the rabbit BBB LAT will also be performed. Because the in vivo Km for the BBB LAT markedly differs among species (i.e. human BBB < rat BBB < rabbit BBB), comparison of the predicted amino acid sequence of BBB LAT from these 3 species will provide insight into the amino acids comprising the active site of the LAT protein, which will be confirmed by site-directed mutagenesis studies. These studies will provide new molecular biological information on a crucial transporter at the blood-brain barrier that regulates the supply in the brain of essential amino acids.